1. Field of the Invention
The present invention relates to a DNA analyzing apparatus, a DNA sensor, and an analyzing method which are used to analyze DNAs having different numbers of bases.
2. Description of the Related Art
In recent years, information on biological genes has been utilized in various fields such as medical care and agriculture. It is essential to understand the structures of DNAs in utilizing genetic information. A DNA has two spirally twisted polynucleotide chains each having a base sequence in which four types of bases (adenine: A, guanine: G, cytosine: C, thymine: T) are one-dimensionally arranged. The base of one of the nucleotide chains is bonded to the base of the other on the basis of the complementarity between adenine and thymine and between guanine and cytosine.
To understand the structure of a DNA is to identify its base sequence. DNA microarrays and their reading apparatuses have been developed in order to identify the base sequences of DNAs. In the prior art, a DNA microarray and its reading apparatus are used to identify the base sequences of sample DNAs as describe below.
First, plural types of single stranded probe DNA fragments are prepared which have known base sequences. The plural types of probe DNA fragments are fixed to a solid carrier such as slide glass, as spots so as to be aligned with one another. On the DNA microarray obtained, the plurality of spots are arranged on an array. One spot is a cluster of probe DNA fragments of one type. Different spots have respective base sequences of probe DNA fragments.
Then, a sample DNA extracted from a specimen is denatured into single stranded sample DNA fragments. A fluorescent substance or the like is bonded to each of the denatured sample DNA fragments. In general, plural types of sample DNA fragments are present.
Then, the plural types of sample DNA fragments are added to the DNA microarray. The plural types of sample DNA fragments are then hybridized to their complementary probe DNA fragments. Specifically, certain types of sample DNA fragments are hybridized to complementary ones of the plural types of probe DNA fragments. However, these types of sample DNA fragments are not hybridized to non-complementary probe DNA fragments. Each of the sample DNA fragments is marked with a fluorescent substance. Consequently, probe DNA fragments bonded to the corresponding sample DNA fragments emit fluorescence.
For example, a sample DNA fragment having the base sequence TCGGGAA is bonded to a probe DNA fragment having the base sequence AGCCCTT. Then, the spot including probe DNA fragments of this type emits fluorescence.
Then, the DNA microarray is set in a reading apparatus. The reading apparatus is used to analyze the DNA microarray. The reading apparatus measures the distribution of fluorescence intensities on the DNA microarray. The distribution of fluorescence intensities on the DNA microarray is outputted as a two-dimensional image. A spot in the outputted image which has a high fluorescence intensity indicates that it contains probe DNA fragments having a base sequence complementary to the base sequence of sample DNA fragments. Accordingly, the base sequence of the sample DNA fragments can be determined depending on which spot in the two-dimensional image has a higher fluorescence intensity.
However, with the above DNA microarray, the sample DNA fragments are simply added to the DNA microarray. Consequently, the distribution of the sample DNA fragments is not uniform. That is, the density of sample DNA fragments may be high on some spots on the DNA microarray but may be low on other spots. Therefore, if the probe DNA fragments in a spot are complementary to any of the plural types of sample DNA fragments, the emission luminance of the fluorescence may vary depending on the density of sample DNA fragments on the spot.
To solve this problem, PCT National Publication No. 11-512605 describes a DNA microarray comprising electrodes which are arranged on a substrate in a matrix and to each of which a spot including probe DNA fragments is fixed. In this microarray, a dispersion medium is applied to the entire surface of the substrate so as to cover all the electrodes and spots.
Description will be given below of an identifying method using the DNA microarray described in PCT National Publication No. 11-512605. First, sample DNA fragments are injected into a dispersion medium. A positive voltage is sequentially applied to the electrodes one by one for spot scanning. Sample DNA fragments in the dispersion medium migrate to selected ones of the plurality of electrodes to which the positive voltage has been applied. Accordingly, the density of the sample DNA fragments is high at the spots of the selected electrodes. Such spot scanning sequentially increases the density of the sample DNA fragments in the respective spots one by one. Therefore, the spots that are complementary to the sample DNA fragments have an almost equal fluorescence intensity. As described above, the technique described in PCT National Publication No. 11-512605 utilizes the electrophoresis of the sample DNA fragments through the dispersion medium.
However, with the conventional DNA microarray, if some of the plural types of sample DNA fragments have a complementary base sequence, the sample DNA fragments may be partially hybridized to one another during electrophoresis. Thus, even if the partially hybridized sample DNA fragments migrate to the complementary spot, they are not hybridized to that spot. As a result, the base sequences of the plural types of sample DNA fragments cannot be identified. Therefore, although the whole electrophoresis medium is heated to denature the sample DNA fragments into a single strand state, it is subsequently cooled to provide for hybridization. This is inefficient. Furthermore, it is impossible to hybridize only arbitrary spots.
It is thus an object of the present invention to efficiently hybridize sample DNA fragments even during electrophoresis.